Composition for culturing nk cells and method for culturing nk cells using same

ABSTRACT

Provided are a composition for culturing NK cells, and a method of culturing NK cells using the same. According to an aspect, in culturing NK cells from peripheral blood mononuclear cells, when NK cells are cultured in a medium including the composition for culturing NK cells, the composition including IL-15, IL-18, and IL-27, the NK cells may proliferate in large quantities and activation of NK cells may be promoted. Therefore, when the NK cells are used, cancer cell apoptosis or cancer cell-killing ability may be promoted. Accordingly, the NK cells may be used as an effective adoptive immune cell therapy product in cancer prevention or treatment.

This application is a continuation of U.S. application Ser. No.16/645,215 filed Mar. 6, 2020, allowed, which is a National Stage ofPCT/KR2018/014294 filed Nov. 20, 2018 and claims priority benefits fromKorean Patent Application No. 10-2017-0158577, filed on Nov. 24, 2017,the entire content of which is fully incorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates to a composition for culturing naturalkiller (NK) cells and a method of culturing NK cells using the same.

BACKGROUND ART

Current cancer therapies generally include surgery, radiation therapy,chemotherapy, etc., which are used alone or in combination depending onthe type and stage of cancer. However, these therapies are accompaniedby significant adverse side effects and pain in patients. The bestcancer therapy is a method of selectively killing only cancer cellswithout damaging normal cells. However, the existing therapies causesome damage to normal cells. Recently emerging cancer immunotherapy is atherapy that more specifically removes cancer cells while minimizingdamage to normal cells by utilizing the body's own immune system.Various sub-fields (antibody therapy, immune cell therapy, viralimmunotherapy, nanotechnology for immunotherapy, etc.) have beenactively studied. Among them, immune cell therapy is a method oftreating cancer by increasing the number of natural killer (NK) cells,natural killer T cells, T cells, B cells, dendritic cells, etc. inlymphocytes obtained from a patient's blood, enhancing their function invitro, and then returning them to the body. Such therapies using immunecells exhibit good effects in immune response-modulating treatment, andare considered to be excellent in terms of toxicity and safety. Amongthe immune cells, NK cells are important cells responsible for innateimmunity. Unlike T cells, NK cells mature in liver and bone marrow. Inparticular, NK cells have functions of identifying and killing abnormalcells such as virus-infected cells or tumor cells. Over the last decade,tumor immunotherapy using patients' immune systems has been steadilydeveloped, and ‘cell therapy products’ using the same have beencommercialized. Cell therapy products are defined as medical productsused for treatment, diagnosis, or prevention through a series of actionsthat change biological characteristics of cells using a method ofpropagating and selecting autologous, allogeneic, or xenogeneic cells invitro (Korea Food & Drug Administration Notification No. 2003-26 Article2).

There is a need for a method of proliferating NK cells in largequantities and a method of culturing NK cells with enhanced activity,which are capable of solving the problem of immune rejection andproviding personalized cell therapy as cell therapy for treatingintractable diseases such as cancer.

DESCRIPTION OF EMBODIMENTS Technical Problem

An aspect provides a composition for culturing natural killer (NK)cells.

Another aspect provides a method of culturing NK cells using thecomposition for culturing NK cells.

Solution to Problem

An aspect provides a composition for culturing natural killer (NK)cells, the composition including IL-15, IL-18, IL-27, or a combinationthereof.

The composition for culturing NK cells may further includeinsulin-transferrin-selenium (ITS).

NK cells are large granular lymphocytes (LGL) which is a type oflymphocytes. They have excellent ability to kill infected virus andtumor cells, and have a characteristic of not killing most normal cells.Thus, NK cells play an important role in the early stages of viralinfection or tumorigenesis before large quantities of active cytotoxic Tlymphocytes are produced. For example, when NK cells are in contact withtarget cells, some molecules lyse the cells by creating pores in themembrane of the target cells while other molecules enter the targetcells and increase fragmentation of nuclear DNA, leading to necrosis,apoptosis, or programmed cell death.

As a result, NK cells are able to lyse specific virus-infected cells andcancer cells in the absence of prior stimulation. Unlike cytotoxic Tlymphocytes which recognize specific antigens through TCR expression, NKcells lack antigen-specific receptors. NK cells express a killer cellimmunoglobulin-like receptor (KIR) that binds with MHC class I of normalcells. Binding of KIR with MHC class I produces intracellular signalingthat induces inhibition of specific transcription factors. As a result,NK cell activation, and target cell lysis and disruption are inhibited.Virus-infected cells or cancer cells have significantly reduced numbersof MHC class I molecules on their surfaces. Thus, when such cellsencounter NK cells, they fail to effectively engage KIRs and thereforeare exposed to NK cell-mediated cytotoxicity and finally lysed.

The NK cells may be derived from, for example, a mammal, a human, amonkey, a pig, a horse, a cow, a sheep, a dog, a cat, a mouse, a rabbit,etc. The NK cells may be obtained from a normal person or a cancerpatient. The NK cells may be isolated from blood or peripheral bloodmononuclear cells (PBMCs). A method of isolating blood, a method ofisolating PBMCs therefrom, or a method of isolating NK cells therefrommay be performed by a known method.

The composition may include IL-15, IL-18, IL-27, or a combinationthereof, in which IL-15 may directly promote growth and differentiationof NK cells through IL-15 receptor expressed in NK cells, IL-18 mayinduce interferon gamma production of NK cells and may enhancecytotoxicity, and IL-27 may regulate the immune response, may enhance asurvival rate of NK cells, and may enhance expression of interferongamma.

The composition may further include ITS, in which ITS may improve NKcell proliferative capacity of the composition for culturing NK cells,the composition including IL-15, IL-18, IL-27, or a combination thereof.

In an NK cell culture medium, a concentration of IL-15 may be about 0.1ng/ml to about 1000 ng/ml, about 0.2 ng/ml to about 500 ng/ml, about 0.5ng/ml to about 200 ng/ml, about 1.0 ng/ml to about 100 ng/ml, about 2.0ng/ml to about 50 ng/ml, or about 5.0 ng/ml to about 20 ng/ml, aconcentration of IL-18 may be about 0.25 ng/ml to about 2500 ng/ml,about 0.5 ng/ml to about 1250 ng/ml, about 1.25 ng/ml to about 500ng/ml, about 2.5 ng/ml to about 250 ng/ml, about 5.0 ng/ml to about 125ng/ml, about 12.5 ng/ml to about 50 ng/ml, and a concentration of IL-27may be about 0.20 ng/ml to about 2000 ng/ml, about 0.40 ng/ml to about1000 ng/ml, about 1.0 ng/ml to about 400 ng/ml, about 2.0 ng/ml to about200 ng/ml, about 4.0 ng/ml to about 100 ng/ml, about 10.0 ng/ml to about40 ng/ml.

The composition may further include IL-7, IL-21, or a combinationthereof. IL-21 may induce maturation of NK cell precursors from bonemarrow, and in particular, may increase effector functions such ascytokine production capacity and cell killing capacity of NK cells. IL-7may induce maturation of NK cell precursors, and may regulateproliferation of T cells. However, even though the composition does notinclude IL-7, IL-21, or a combination thereof, it may promoteproliferation or activation of NK cells.

A concentration of IL-7 may be about 0.05 ng/ml to about 500 ng/ml,about 0.10 ng/ml to about 250 ng/ml, about 0.25 ng/ml to about 100ng/ml, about 0.5 ng/ml to about 50 ng/ml, about 1.0 ng/ml to about 25ng/ml, about 2.5 ng/ml to about 10 ng/ml, and a concentration of IL-7may be about 0.05 ng/ml to about 500 ng/ml, about 0.10 ng/ml to about250 ng/ml, about 0.25 ng/ml to about 100 ng/ml, about 0.50 ng/ml toabout 50 ng/ml, about 2.5 ng/ml to about 10 ng/ml.

The medium refers to a material capable of supporting growth andsurvival of cells in vitro. The medium is not particularly limited, aslong as it may be used in cell culture, and the medium may include, forexample, one or more selected from the group consisting of Dulbecco'sModified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), BasalMedium Eagle (BME), RPMI 1640, F-10, F-12, DMEM/F12, Minimal EssentialMedium-α (MEM-α), Glasgow's Minimal Essential Medium (G-MEM), Iscove'sModified Dulbecco's Medium (IMDM), MacCoy's 5A medium, AmnioMax completemedium, AminoMax II complete medium, Endothelial Basal Medium (EBM)medium, and Chang's Medium.

The NK cells may have surface antigen characteristics of CD3⁻ and CD56⁺.

The culture may be for proliferation or activation of the NK cells. Theproliferation of the NK cells means an increase in the number of cells,and may be used interchangeably with growth. The activation of the NKcells means that the above-described NK cells perform their functions,and may have surface antigen characteristics of CD226⁺, CD69⁺, CD14⁻,CD19⁻, CD16⁺, or a combination thereof.

The surface antigen characteristics have the same meaning asimmunological characteristics, and may be identified by observing cellsurface markers (e.g., staining of cells with tissue-specific orcell-marker specific antibody) using a technique such as flow cytometryor immunocytochemistry, or by observing cell surface marker using anoptical microscope or a confocal microscope, or by measuring changes ingene expression patterns using a technique known in the art, such aspolymerase chain reaction (PCR) or gene expressing profiling.

The “positive or +” may mean that, with regard to a cell marker, themarker is present in a large quantity or at a high concentration, ascompared with that of other reference cells. A marker exists inside acell or on the surface of the cell, and therefore, when the marker maybe used to distinguish the cell from one or more other types of cells,the cell may be positive for the marker. The positive may also mean thatthe cell has the marker with a signal intensity higher than a backgroundsignal, for example, in an amount sufficient to be signal-detected by acell measuring device. For example, when cells may be detectably labeledwith a CD56-specific antibody, and signals from the antibody aredetectably higher than those of a control (e.g., background), the cellmay be represented by “positive for CD56” or “CD56⁺”. The “negative or−” may mean that even though a specific cell surface marker-specificantibody is used, it is not detectable, as compared with a background.For example, cells may not be detectably labeled with a CD3-specificantibody. The cell may be represented by “negative for CD3” or “CD3⁻”.

Another aspect provides a method of culturing NK cells, the methodincluding culturing NK cells in a medium including a composition forculturing NK cells, the composition including IL-15, IL-18, and IL-27.

The culturing method may be a method of proliferating NK cells in largequantities or a method of activating NK cells.

The method may further include, before culturing, obtaining peripheralblood mononuclear cells (PBMCs) from peripheral blood; and isolating NKcells from the obtained PBMCs.

The above process may further include removing cells having a surfaceantigen characteristic of CD3⁺.

A method of isolating blood, a method of isolating PBMCs therefrom, amethod of isolating NK cells therefrom, and a method of isolating orremoving cells having specific surface antigen characteristics may beperformed by a known method of using specific antibodies, etc.

According to the method, even when NK cells are cultured for a long timeor sub-cultured several times, NK cells have excellent proliferativecapacity, as compared with groups cultured with other cytokines, andtherefore, NK cells may be obtained in large quantities.

According to the method, NK cells cultured by adding the composition mayhave increased interferon-gamma (IFN-gamma) secretion, as compared withgroups cultured with other cytokines. Further, NK cells cultured byadding the composition may have increased cytotoxicity against cancercells and may promote apoptosis of cancer cells, as compared with groupscultured with other cytokines.

The culture may be performed for about 7 days to about 2 days, about 7days to about 30 days, about 14 days to about 30 days, about 15 days toabout 27 days, about 16 days to about 26 days, about 17 days to about 25days, about 18 days to about 24 days, about 19 days to about 23 days, orabout 20 days to about 22 days.

Still another aspect provides NK cells prepared by the method ofculturing NK cells.

Still another aspect provides a composition for preventing or treatingcancer, the composition including NK cells prepared by the method ofculturing NK cells.

Still another aspect provides use of the NK cells prepared by the methodof culturing NK cells in preparing a pharmaceutical composition orformulation for preventing or treating cancer.

Still another aspect provides use of the NK cells prepared by the methodof culturing NK cells in preparing a medical product for preventing ortreating a disease, for example, cancer. The cancer may be solid cancer,lung cancer, liver cancer, breast cancer, uterine cancer, blood cancer,etc., but is not limited thereto. In cancer patients, the NK cells arereported to be closely related with development of diseases, such aslung cancer (Carrega P, et al., Cancer, 112, 863-875, 2008), livercancer (Jinushi M, et al., J Hepatol., 43, 1013-1020, 2005), breastcancer (Bauernhofer T, et al., Eur J Immunol., 33, 119-124, 2003.),uterine cancer (Mocchegiani E., et al., Br j Cancer., 79, 244-250,1999), blood cancer (Tajima F., et al, Lekemia, 10, 478-482, 1996), etc.

The composition may include a pharmaceutically acceptable carrier. Inthe composition, the “pharmaceutically acceptable carrier” refers to amaterial, generally, an inert material used in combination with anactive ingredient to aid application of the active ingredient. Thecarrier may be an excipient, a disintegrant, a binder, a lubricant, adiluent, or a combination thereof. The excipient may be microcrystallinecellulose, lactose, low-substituted hydroxycellulose, or a combinationthereof. The disintegrant may be sodium starch glycolate, anhydrousdibasic potassium phosphate, or a combination thereof. The binder may bepolyvinylpyrrolidone, low-substituted hydroxypropyl cellulose,hydroxypropyl cellulose, or a combination thereof. The lubricant may bemagnesium stearate, silicon dioxide, talc, or a combination thereof.

Still another aspect provides a method of treating cancer, the methodincluding administering, to an individual, a therapeutically orpharmaceutically effective amount of the NK cells prepared by the methodof culturing NK cells.

The term “administering” refers to introduction of a predeterminedsubstance into an individual in any appropriate manner, and with regardto the administration route, the substance may be administered throughany general route as long as it may reach a target tissue. Theadministration route may be intraperitoneal administration, intravenousadministration, intramuscular administration, subcutaneousadministration, intradermal administration, oral administration, topicaladministration, intranasal administration, intrapulmonaryadministration, or rectal administration, but is not limited thereto. Inaddition, administration may be performed by any apparatus capable ofmoving to a target cell. An administration dose may be appropriatelyselected according to the type of cancer, the administration route, apatient's age and gender, and severity of a disease, but for an adult,it may be administered at a dose of about 1×10⁶ to about 1×10¹¹ cells onaverage.

The “therapeutically effective amount” means an amount sufficient toexhibit a therapeutic effect when administered to an individual or acell in need of treatment. The “treatment” means treating a disease ormedical condition in an individual, for example, a mammal, including ahuman, and the treatment includes: (a) prevention of generation of adisease or medical symptoms, i.e., preventive treatment of a patient;(b) relief of a disease or medical symptoms, i.e., removal or recoveryof a disease or medical symptoms in a patient; (c) suppression of adisease or medical symptoms, i.e., slowing or stopping progression of adisease or medical symptoms in an individual; or (d) alleviation of adisease or medical symptoms in an individual.

Advantageous Effects of Disclosure

According to an aspect, in culturing NK cells from peripheral bloodmononuclear cells, when NK cells are cultured in a medium including acomposition for culturing NK cells, the composition including IL-15,IL-18, and IL-27, the NK cells may proliferate in large quantities andactivation of NK cells may be promoted. Therefore, when the NK cells areused, cancer cell apoptosis or cancer cell-killing ability may bepromoted, and therefore, the NK cells may be used as an effectiveadoptive immune cell therapy product in cancer prevention or treatment.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows results of counting the numbers of PBMCs and NK cellsisolated from healthy normal individuals;

FIG. 2A shows FACS results of analyzing phenotypes of NK cellsimmediately (DO) after isolating the NK cells from PBMCs isolated fromhealthy normal individuals and 21 days (D21) after culturing the NKcells;

FIG. 2B shows results of analyzing distributions of CD3⁻CD56⁺ NK cellsimmediately (DO) after isolating PMBCs and NK cells from healthy normalindividuals and 21 days (D21) after culturing the PMBCs and NK cells;

FIG. 2C shows results of calculating the purity of NK cells immediately(DO) after isolating the NK cells from PBMCs isolated from healthynormal individuals and 21 days (D21) after culturing the NK cells;

FIG. 3A shows photographs showing results of culturing NK cells for 3days (Day 3), 7 days (Day 7), 17 days (Day 17), and 21 days (Day 21) inthe composition for culturing NK cells of the present disclosure;

FIG. 3B shows graphs showing the numbers of NK cells cultured for 3 days(Day 3), 7 days (Day 7), 17 days (Day 17), and 21 days (Day 21) in thecomposition for culturing NK cells of the present disclosure;

FIG. 3C shows a graph showing proliferation of NK cells on day 0 (DO),day 3 (D3), day 13 (D13) and day 21 (D21) according to cytokinecocktails;

FIG. 4 shows results of examining effects of the composition forculturing NK cells of the present disclosure on NK cell activity;

FIG. 5A shows photographs of NK cells cultured for 21 days in a culturemedium containing IL-2; IL-15 and IL-18 (IL-15/18); or IL-15, IL-18, andIL-27 (IL-15/18/27), with ITS (+ITS) or without (−ITS);

FIG. 5B shows a graph showing the number and proliferation of NK cellsas a result of culturing using 6-well plates for 0-5 days, culturingusing T25 flasks for 5-12 days, and then culturing for 21 days aftertransferring to T75 flasks on day 21;

FIG. 5C shows results of examining cell viability of NK cells when theNK cells are cultured as in FIG. 5B;

FIG. 6A shows a photograph showing morphology of NK cells when culturedusing a mixed culture medium of cytokines IL-15, IL-18, and IL-27 andITS;

FIG. 6B shows graphs showing a growth curve and proliferation of NKcells when cultured using a mixed culture medium of cytokines IL-15,IL-18, and IL-27 and ITS;

FIG. 6C shows a graph showing viability of NK cells when cultured usinga mixed culture medium of cytokines IL-15, IL-18, and IL-27 and ITS;

FIG. 7 shows results of examining cancer cell cytotoxicity of NK cellscultured by adding the composition for culturing NK cells of the presentdisclosure;

FIG. 8 shows a graph showing ELISA results of examining IFN-γ secretedfrom NK cells on day 7 (D7), day 14 (D14), and day 21 (D21) followingculture;

FIG. 9 shows results of examining cancer cell cytotoxicity of NK cellscultured by adding the composition for culturing NK cells of the presentdisclosure;

FIG. 10 shows results of examining activity, expression patterns, andexpression levels of caspase-3 in cancer cells which were co-culturedwith NK cells cultured by adding the composition for culturing NK cellsof the present disclosure; and

FIG. 11 shows images of expression levels and expression locations ofcaspase-3 in cancer cells which were co-cultured with NK cells culturedby adding the composition for culturing NK cells of the presentdisclosure.

MODE OF DISCLOSURE

Hereinafter, the present disclosure will be described in more detailwith reference to exemplary embodiments. However, these exemplaryembodiments are only for illustrating the present disclosure, and thescope of the present disclosure is not limited to these exemplaryembodiments.

Example 1: Isolation of Natural Killer (NK) Cells and Examination ofProliferative Capacity of NK Cells, Activity of NK Cells, andCytotoxicity of NK Cells against Cancer Cells, After Culturing NK Cellsusing Composition for Culturing NK Cells

1. Isolation of NK Cells and Culturing of NK Cells Using Composition forCulturing NK Cells

(1.1) Selection of Research Subjects, Isolation of Blood and PBMCs, andIsolation of NK Cells

Healthy males and females aged 20 to 65 years were subjects who agreedto blood collection for this study, and subjects eligible to participatein this study were selected based on health questionnaire, body weight,and vital signs. Subjects eligible to participate in the study wereselected by the following criteria.

-   -   1) Those who do not have the following exclusions through health        questionnaire        -   Those with a history of cardiovascular diseases such as            hypertension, etc., kidney disease, diabetes, and cancer        -   Those who refuse blood transfusion for religious reasons,            etc.        -   Pregnant women    -   2) Males having body weight of 50 kg or more and females having        body weight of 45 kg or more    -   3) Those who satisfy the following vital signs        -   Blood pressure (mmHg): systolic pressure of 90 to 179 and            diastolic pressure of less than 100        -   Body temperature (° C.): 37.5° C. or less        -   Pulse (beats/min): 50 to 100

On the day of visit, a total of 100 ml of blood was collected in a tubecontaining heparin once from those selected as research subjects,respectively.

From the collected blood, peripheral blood mononuclear cells (PBMCs)were isolated using Ficoll-Paque (GE Healthcare, 17-1440-02) by thefollowing method. The collected whole blood was diluted 1:1 withphosphate buffered saline (PBS) (pH 7.4, Thermo Fisher Scientific), andthe diluted blood was carefully added to the top of Ficoll.Subsequently, centrifugation was performed at 2500 rpm and 25° C. for 22minutes to isolate PBMCs. The isolated PBMCs were washed with PBS.Thereafter, PBMCs without any treatment were stored before isolation ofNK cells. Further, plasma obtained during the isolation process was alsocollected and stored.

From the isolated PBMCs, NK cells were isolated using an NK cellisolation kit (Miltenyi Biotec, 130-092-657) and CD3⁺ magnetic beads(Miltenyi Biotec) by the following method, and CD3⁺ cells were removed.MACS running buffer (PBS, 2 mM EDTA, 0.5% BSA) was added to the isolatedPBMCs in a volume of 40 μl per 1×10⁷ cells to suspend PBMC cell pellets,and then CD3⁺ magnetic beads were added in a volume of 20 μl per 1×10⁷cells and allowed to react at 4° C. for 10 min. Subsequently, the cellswere washed with MACS buffer, and then CD3⁻CD56⁺ NK cells were recoveredusing a MACS cell separator (Miltenyi Biotec).

FIG. 1 and Table 1 show results of counting the numbers of PBMCs and NKcells isolated from healthy normal individuals. FIG. 1 shows results ofcounting the number of PBMCs isolated from healthy normal individualsand the number of NK cells isolated from healthy normal individuals. Asshown in FIG. 1 and Table 1, distribution of the CD3⁻CD56⁺ NK cells inPBMCs in the blood was about 8% to about 20% on average. There was nodifference according to gender, and there was no statisticallysignificant increase or decrease in NK cells according to age.

TABLE 1 Number of healthy Age normal Average (Mean ± SD) Gender (years)individuals PBMC(×10⁸) NK(×10⁷) Female 20-29 5 2.564 ± 0.952 2.526 ±1.259 30-39 5 1.634 ± 0.461 1.220 ± 0.139 40 2 1.952 ± 1.294 2.455 ±0.955 Total 12 Male 20-29 2 2.385 ± 0.587 2.425 ± 0.955 30-39 9 1.965 ±0.579 2.294 ± 1.264 40-49 3 1.237 ± 0.220 2.100 ± 0.383 Total 14

Subsequently, whether NK cells in the isolated PBMCs and the collectedNK cells had CD3⁻CD56⁺ characteristics was examined by fluorescenceactivated cell sorting (FACS, BD FACSCalibur).

FIG. 2 shows FACS results of analyzing phenotypes of NK cells in PBMCsisolated from healthy normal individuals and the collected NK cells.FIG. 2A shows the results of analyzing phenotypes of NK cellsimmediately (DO) after isolating the NK cells and 21 days (D21) afterculturing the NK cells. FIG. 2B shows results of analyzing distributionsof CD3⁻CD56⁺ NK cells immediately (DO) after isolating PMBCs and NKcells and 21 days (D21) after culturing the PMBCs and NK cells.

Referring to FIG. 2 , NK cells (CD3⁻CD56⁺) collected from healthy normalindividuals (#8) had purity of 95% or more, and distributions of Tcells, B cells, and monocytes were about 1% to about 2%. NK cells (D21)after culture for 21 days had purity of 98% or more.

(1.2) Culture of NK Cells Using Composition for Culturing NK Cells

NK cells obtained in (1) were added at a density of 1×10⁵ cells/ml ineach well of a 12- or 24-well tissue culture plate, and CellGro®serum-free medium (CellGenix, USA), 10% human serum (Sigma Aldrich,USA), 10,000 U/mL penicillin/streptomycin (Pen/Strep) (Gibco/LifeTechnologies, Carlsbad, CA), cytokine (IL-15, IL-27, 1-100 ng/ml;Peperotech, Inc. NJ, USA; IL-18, 1-100 ng/ml; R&D Systems, Inc., MN,USA), and ITS (Insulin-Transferrin-Selenium-G Supplement 100λ, Gibco™)were added thereto, followed by culturing in an incubator at 37° C. and5% CO₂ for 21 days.

In detail, 4 sets of cytokine combination treatment groups including acombination treatment group 1-1: IL-7 (5 ng/ml), IL-15 (10 ng/ml), IL-18(25 ng/ml), IL-21 (5 ng/ml), and IL-27 (20 ng/ml), a combinationtreatment group 1-2: IL-15 (10 ng/ml), IL-18 (25 ng/ml), IL-21 (5ng/ml), and IL-27 (20 ng/ml), a combination treatment group 1-3: IL-15(10 ng/ml), IL-18 (25 ng/ml), and IL-27 (20 ng/ml), and a combinationtreatment group 1-4: IL-15 (10 ng/ml) and IL-18 (25 ng/ml) were mixed ina fresh medium, respectively, and then, once every 2 to 3 days, which isthe time to replace the medium, the medium was added to the NK cellsduring culture, and cultured. Compositions of the media are the same asdescribed above, except for cytokines.

FIG. 2C shows results of analyzing the purity of NK cells immediately(DO) after isolating the NK cells and 21 days (D21) after culturing theNK cells. According to FIG. 2C, NK cells collected from the healthynormal individuals (#8) were found to have the purity of about 95% ormore, even after cultured by adding the composition for culturing NKcells of the combination treatment group 1-3.

2. Examination of Proliferative Capacity of NK Cells and Activity of NKCells after Cultured by Adding Composition for Culturing NK Cells

(2.1) Examination of Proliferative Capacity of NK Cells after Culturedby Adding Composition for Culturing NK Cells

The number NK cells proliferated during culture as in (1.2) wasincreased, starting from 1×10⁵ cells/ml to 1×10⁶ cells/ml of NK cells ina 6-well tissue culture plate to a large amount in T25, T75, and BAG(NIPRO cell culture bags, A-1000NL, A-350NL, Funakoshi Co, Ltd.) atintervals of 2 days to 3 days. Viability was determined using ahemocytometer after staining the propagated NK cells with a trypan bluestaining agent (Thermo Fisher Scientific, USA).

FIG. 3 shows results of examining the effect of the composition forculturing NK cells of the present disclosure on proliferation andviability of NK cells. FIG. 3A shows photographs showing results ofculturing NK cells for 3 days (Day 3), 7 days (Day 7), 17 days (Day 17),and 21 days (Day 21) in the composition for culturing NK cells of thepresent disclosure. As shown in FIG. 3A, when NK cells were cultured(stimulated) by adding the combination treatment group 1-3 among thecompositions for culturing NK cells of the present disclosure, primaryNK cells grew continuously while forming clusters, and their cellviability was as high as about 85% to about 90% on average for 21-dayculture. FIG. 3B shows graphs showing the numbers of NK cells. As shownin FIG. 3B, the number of the NK cells of healthy normal individuals(#12) showed about 26 times increase from about 6×10⁶ cells to 1.58×10⁸cells, after cultured for 21 days by adding the combination treatmentgroup 1-3, as compared with those before culture. Further, the number ofthe NK cells of healthy normal individuals (#2) showed about 40 timesand about 53.3 times increase, after cultured by adding the combinationtreatment group 1-2 and the combination treatment group 1-3,respectively. FIG. 3C shows a graph showing proliferation of NK cells onday 0 (D0), day 3 (D3), day 13 (D13) and day 21 (D21) according tocytokine cocktails. As shown in FIG. 3C, the proliferation of NK cellsshowed the significantly increased number of cells, when cultured for 21days by adding the combination treatment group 1-3. Small differencesbetween normal individuals were observed.

(2.2) Examination of Activity of NK Cells after Cultured by AddingComposition for Culturing NK Cells

During culture as in (1.2), differences in activity of NK cells andreceptor expression were examined.

During culture of NK cells, NK cells were collected, and 1×10⁵ NK cellswere dispensed in a FACS tube (Falcon® 5 mL Round Bottom PolystyreneTest Tube). An antibody of a target gene was added to the FACS tube, andallowed to react for 30 min. Thereafter, washing was performed usingFACS buffer, and surface antigen characteristics were analyzed usingfluorescence-activated cell sorting (FACS, BD FACSCalibur™). FlowJoprogram was used for data analysis.

Target gene Company Cat. CD3 APC BD 555342 CD3 PE Invitrogen 12-0038-42CD56 FITC BD 562794 CD56 PE BD 555516 Activating receptor CD314/NKG2D PEThermo 12-5878-42 CD335 PE BD 557991 CD336 PE BD 558563 CD337 PE BD558407 CD226 (DNAM) PE BD 559789 Inhibitory receptor NKG2A PE R&DFAB1059P KIR2DL4 (CD158d) PE R&D FAB2238P-100 KIR2DL5A (CD158f1) PEOrigene AM26776RP-N KIR2DL1/158a FITC BD 556062 KIL2DL2/3 158b FITC BD559784 KIR3DL1 (CD158e1, NKB1) FITC BD 555966 KIR3DL2 PE R&DFAB2878P-100 KIR3DL3 (CD158z) Alexa 647 R&D FAB8919R-025 Surface markerCD14 PE BD 555398 CD19 FITC BD 555412 CD69 PE Invitrogen MHCD6904/I701544A CD96 PE BD 562379 CD16 PE Thermo MHCD1604

FIG. 4 shows results of examining effects of the composition forculturing NK cells of the present disclosure on NK cell activity.Referring to FIG. 4 , an expression level of CD226 which is one ofactivating receptors on NK cells was increased from about 12.3% to about95.7%, after cultured by adding the combination treatment group 1-3(D21), as compared with those before culture (DO). Further, anexpression level of CD69 which is an activation index was increased fromabout 3.3% to about 91.4%, after cultured by adding the combinationtreatment group 1-3 (D21), as compared with those before culture (DO).It was found that a large quantity of NK cells may be induced andproliferated only by using a small number of cytokines without feedercells.

(2.3) Examination of NK Cell Proliferation Effect of ITS Addition

It was examined whether NK cell proliferation effect may be increased byadding insulin-transferrin-selenium (ITS) during culture of NK cells. NKcells were cultured for 21 days in three different culture media, eachcontaining IL-2; IL-15 and IL-18; or IL-15 and IL-18 and IL-27 with orwithout ITS (Insulin-Transferrin-Selenium-G Supplement 100λ, Gibco™).The results of culture are shown in FIG. 5 .

FIG. 5A shows photographs of NK cells cultured for 21 days in a culturemedium containing IL-2; IL-15 and IL-18 (IL-15/18); or IL-15, IL-18 andIL-27 (IL-15/18/27) with ITS (+ITS) or without (−ITS).

As shown in FIG. 5A, when ITS was added to the culture medium containingIL-2; IL-15 and IL-18; or IL-15 and IL-18 and IL-27, NK cells were moreactively proliferated to form clusters, indicating a synergistic effect.

FIG. 5B shows a graph showing the number and proliferation of NK cellsas a result of culturing using 6-well plates for 0-5 days, culturingusing T25 flasks for 5-12 days, and then culturing for 21 days aftertransferring to T75 flasks on day 21.

As shown in FIG. 5B, in the NK cell proliferation, improvement in NKcell proliferation by ITS was observed. When treated with onlyIL-15/IL-18/IL-27, 13.76 times increase (2.064×10⁷) was observed, ascompared with the initial. In contrast, when cultured with a mixture ofIL-15/IL-18/IL-27 and ITS, 27.88 times increase (4.1825×10⁷) wasobserved.

FIG. 5C shows results of examining cell viability when the cells werecultured as above.

As shown in FIG. 5C, both two cells showed viability of 95% or more andcluster formation. Therefore, it was found that use of ITS does notinfluence cell viability.

Taken together, it was confirmed that addition of ITS may furtherimprove the NK cell proliferation effect in the composition of culturingNK cells of the present disclosure.

(2.4) Examination of NK Cell Culture in Culture Bag

It was examined whether the method of culturing NK cells as confirmedabove may be applied to mass-production of NK cells. In detail, NK cellsobtained from the same subjects were activated using IL-15/18/27 earlyon days 0-7, and then ITS was added to the existing NK cell culturemedium, followed by culture in a T25 plate. On days 7-12, the cells weretransferred to several T25 plates, followed by culture. After 12-14 daysof culture, the cells were transferred to a culture bag, followed byculture for 21 days.

FIG. 6 shows a photograph of NK cells cultured in the culture bag.

FIG. 6A shows a photograph showing morphology of NK cells when culturedusing a mixed culture medium of cytokines IL-15, IL-18, and IL-27 andITS.

FIG. 6B shows graphs showing a growth curve and proliferation of NKcells when cultured using a mixed culture medium of cytokines IL-15,IL-18, and IL-27 and ITS.

As shown in FIGS. 6A and 6B, NK cells also formed clusters in theculture bag, as in the culture of using the plate. Further, cell growthwas increased to 2.84×10⁸, which was about 31.53 times, as compared withthe initial number of NK cells, indicating continuous growth for 21days.

FIG. 6C shows a graph showing viability of NK cells when cultured usinga mixed culture medium of cytokines IL-15, IL-18, and IL-27 and ITS.

As shown in FIG. 6C, high cell variability of 98% or more was observed.

Therefore, when NK cells were cultured using the composition forculturing NK cells according to the present disclosure, the NK cellproliferation effect was observed not only in the plate but also in theculture bag, indicating that the composition may also be applied tomass-production.

3. Examination of Cytotoxicity of NK Cells Against Cancer Cells UsingComposition for Culturing NK Cells

(3.1) Examination of Cancer Cell Cytotoxicity of NK Cells Cultured byAdding Composition for Culturing NK Cells 1

(3.1.1) Cytotoxicity Assay

Cytotoxicity assay was performed for K562 cell (human chronicmyelogenous leukemia cell line), which is generally used for measuringNK cell activity because of high sensitivity to NK cells, using aCytotTox-Glo™ cytotoxicity assay kit of Promega. This assay is a methodof measuring an enzyme released as a result of cell membrane damage, inwhich enzymatic reaction of dead cells may be measured by examining aluminogenic peptide substrate(alanyl-alanyl-phenylalanyl-aminoluciferin, AAF-Glo Substrate).

K562 cells were dispensed at a density of 1×10⁴ cells in each well of a96-well plate coated with poly-D-lysine, and NK92 and NK cells forcytotoxicity assay were added thereto at an E:T ratio of 0:1, 1.25:1,2.5:1, 5:1, or 10:1, and allowed to react for 4 hr. Thereafter, 50 μL ofCytotTox-Glo™ cytotoxicity assay reagent was added and allowed to reactat room temperature for 15 min, and then enzymatic activity of deadcells was measured using a luminometer. Subsequently, 50 μL of lysisbuffer was added and allowed to react for 15 min, and then the totalnumber of cells was examined. A percentage of the dead cells to thetotal number of cells was calculated to analyze cytotoxicity. At thistime, the obtained data were analyzed using Microsoft Excel and GraphPadPrism 5.0.

FIG. 7 shows results of examining cancer cell cytotoxicity of NK cellscultured by adding the composition for culturing NK cells of the presentdisclosure. Referring to FIG. 7 , when cells were cultured using IL-2alone, IL-15 alone, IL-15 and IL-18 (IL-15/18), IL-15 and IL-27(IL-15/27), IL-18 and IL-27 (IL-18/27), or IL-15, IL-18 and IL-27(IL-15/18/27), remarkably increased NK cytotoxicity was observed on day7 (D7), day 14 (D14), and day 21 (D21) in NK cells cultured using acombination of two or more cytokines, as compared with those culturedusing cytokine alone. In particular, when cultured using a combinationof IL-15, IL-18, and IL-27, the highest cytotoxicity was maintained evenduring long-term (21 days) culture of NK cells. In a comparativeexperiment using an NK-92 cell line, the cytotoxicity assay value (RLU)of the NK cells was also 2 time or higher than that of NK-92. Theseresults indicate that cancer cell cytotoxicity of NK cells stimulatedwith a combination of IL-15, IL-18, and IL-27 was further increased, ascompared with those stimulated with IL-2 or IL-15 alone.

Additionally, cytotoxicity was also examined by measuring changes inexpression of IFN-γ secreted by NK cells. In detail, during NK cellculture, 1 ml of NK cell culture medium was collected every 7 days. Inthe collected culture media, IFN-γ expression was measured using anenzyme-linked immunosorbent assay (ELISA, R&D, MN, USA), and resultswere analyzed using an ELISA Microplate Reader, and shown in FIG. 8 .

FIG. 8 shows a graph showing ELISA results of examining IFN-γ secretedfrom NK cells on day 7 (D7), day 14 (D14), and day 21 (D21) followingculture.

As shown in FIG. 8 , when treated with IL-2 alone, IL-15 alone, IL-15and IL-18 (IL-15/18), IL-15 and IL-27 (IL-15/27), or IL-15, IL-18, andIL-27 (IL-15/18/27), a high expression level of IFN-γ secreted by NKcells was observed in the IL-15/18/27-treated group, as compared withother cytokine-treated groups.

Therefore, stimulation of NK cells with cytokines effectively activatedimmune cells present in blood, leading to IFN-γ secretion. Further,since this result shows a significant relationship with the cytotoxicityresult of NK cells, measurement of the IFN-γ secretion capacity of NKcells may represent cytotoxicity of NK cells.

(3.2.2) Calcein AM Assay

K562 cells stained with Calcein AM (Thermo Fisher Scientific) weredispensed at a density of 1×10⁵ cells in each well of a 6-well platecoated with poly-D-lysine, and NK92 and NK cells for cytotoxicity assaywere added thereto at an E:T ratio of 0:1, 1.25:1, 2.5:1, 5:1, or 10:1,and co-cultured for 21 days. Thereafter, a Calcein AM release assay wasperformed, and lysis of K562 cells by NK cells was observed under afluorescence microscope (zeiss microscope).

FIG. 9 shows results of examining cancer cell cytotoxicity of NK cellscultured by adding the composition for culturing NK cells of the presentdisclosure. The green-stained areas represent K562 cells. As shown inFIG. 9 , as the ratio of NK cells (effectors) was higher, the number oflive K562 cells was lower. This result suggests that apoptosis of K562cell may be caused by interaction between NK cells and K562 cells.

(3.3) Examination of Cancer Cell Cytotoxicity of NK Cells Cultured byAdding Composition for Culturing NK Cells2.

(3.3.1) Caspase-3 Immunoblot

For in vitro cytotoxicity test of NK cells cultured by adding thecomposition for culturing NK cells of the present disclosure, ovariancancer cells and NK cells were cultured, and then activity andexpression levels of caspase-3 in the ovarian cancer cells wereexamined.

Ovarian cancer cells (A2780, SKOV3) were dispensed at a density of 1×10⁵cells in each well of a 6-well plate coated with poly-D-lysine, and onnext day, 1×10⁵ of NK92 and NK cells of the present disclosure forcytotoxicity assay were cultured with the ovarian cancer cells for 3 hr.Then, the culture medium and NK cells were removed, and only theremaining ovarian cancer cells were collected, followed by cell lysisusing a lysis buffer. The cell lysate was heated at 95° C. for 10 min,and then proteins were isolated by centrifugation at 13,000 rpm for 20min. The obtained proteins were separated using SDS-polyacrylamide gelelectrophoresis (SDS-PAGE), and the separated proteins were transferredonto a polyvinylidene fluoride (PVDF) membrane (EMD Millipore,Billerica, MA, USA). The PVDF membrane was incubated with 5% skim milkto block non-specific antibody binding. Subsequently, the PVDF membranewas reacted with a primary antibody, anti-caspase-3, and anti-actin at4° C. overnight, respectively. On next day, primary antibody-bound PVDFmembrane was reacted with a peroxidase-conjugated secondary antibody atroom temperature for 1 hr. Protein bands were visualized and quantifiedusing an enhanced chemiluminescence (ECL) kit system.

FIG. 10 shows results of examining activity, expression patterns, andexpression levels of caspase-3 in cancer cells which were co-culturedwith NK cells cultured by adding the composition for culturing NK cellsof the present disclosure. With progression of apoptosis, a whole formof caspase-3 was decreased, and a cleaved form thereof was increased. Asshown in FIG. 10 , the cancer cells which were co-cultured with NK cellscultured by adding the composition for culturing NK cells of the presentdisclosure showed increased protein quantity of caspase-3 cleaved form,indicating a significant increase of cytotoxicity against cancer cells.Further, the expression level of caspase-3 cleaved form in cancer cellswhich were co-cultured with NK cells cultured by adding the compositionfor culturing NK cells of the present disclosure showed about 5% toabout 10% increase, as compared with that of cancer cells co-culturedwith NK92 cells.

(3.3.2) Caspase-3 Immunofluorescence Staining

Subsequently, apoptosis progression in cancer cells was examined.

Ovarian cancer cells (A2780, SKOV3) were dispensed on a chamber slidecoated with poly-D-lysine, and on next day, the equal numbers of NK92cells and NK cells of the present disclosure were cultured with theovarian cancer cells for 3 hr, respectively. The culture medium and NKcells were removed, and the remaining ovarian cancer cells weresubjected to immunofluorescence staining. As a primary antibody,anti-caspase3 and anti-actin were used, and as a secondary antibody,Alexa 488 goat anti-rabbit and Alexa 546 goat anti-mouse were used.Actin and DAPI were used as controls. After staining, the slide wasmounted, and observed under a confocal laser-scanning microscope.

FIG. 11 shows images of expression levels and expression locations ofcaspase-3 in cancer cells which were co-cultured with NK cells culturedby adding the composition for culturing NK cells of the presentdisclosure.

As shown in FIG. 11 , the cancer cells which were co-cultured with NKcells cultured by adding the composition for culturing NK cells of thepresent disclosure showed about 50% or more increase in the caspase-3activity, as compared with the control, and about 10% or more increasein the caspase-3 activity, as compared with cancer cells co-culturedwith NK92 cells as another control.

1. A method of treating cancer, the method comprising administering aneffective amount of NK cells prepared by a method of culturing naturalkiller cells (NK cells), wherein the method comprising culturing the NKcells in a composition for culturing NK cells, the compositioncomprising IL-15 in an amount of 5.0 ng/ml to 20 ng/ml, IL-18 in anamount of 0.25 ng/ml to 2500 ng/ml, IL-27 in an amount of 0.20 ng/ml to2000 ng/ml, and insulin transferrin selenium (ITS).
 2. The method ofclaim 1, wherein the composition further comprises IL-7, IL-21, or acombination thereof.